TWI354792B - The method of stirring solution - Google Patents

Abstract

The method of stirring a solution according to the present invention is a method of stirring a solution comprising contacting a selective binding substance immobilized on the surface of a carrier with a solution containing an analyte substance reactive with the selective binding substance, and mixing the fme particles or air bubbles into the solution containing an analyte substance, and moving the fine particles or air bubbles without allowing contact thereof with the selective binding substance-immobilized surface. The present invention provides a stirring method that accelerates the reaction of a carrier-immobilized selective binding substance with an analyte substance and detects a trace amount of analyte at high signal intensity and high S/N ratio. The present invention enables diagnosis and examination in the clinical setting by using a selective binding substance-immobilized carrier such as DNA chip.

Description

1354792 IX. Description of the invention: [Technical field to which the invention pertains]

The present invention relates to a solution agitation method in which a carrier to which a substance to be selectively bound and a substance to be tested is selectively bound (referred to as "selective binding substance" in the present specification) is contacted with a solution containing the substance to be tested, A method of stirring a solution containing a test substance when the selective binding substance immobilized on the carrier reacts with the test substance. More specifically, it relates to a method of stirring a solution containing a test substance for promoting a reaction between a selective binding substance immobilized on a carrier and a test substance. [Prior Art] Research on the analysis of genetic information of various organisms is underway. The information on the majority of genes and their base sequences, or the proteins decoded by the gene sequences and the sugar chains produced by the secondary proteins, which are represented by human genes, are rapidly being solved one by one. The role of polymers such as genes, proteins, and sugar chains whose sequences have been solved can be investigated by various methods. The nucleic acid is mainly based on the complementarity of various nucleic acids/nucleic acids such as northern blotting or southern pulverization (s 〇uthernb 1 〇11 ing ), and can investigate the relationship between various genes and their biological functions. . Proteins can be investigated for protein/protein reactions represented by western blotting, and the role and performance of proteins can be investigated. In recent years, as a method of analyzing the expression of a large number of genes, a new analysis method called D N A microarray method (D N A wafer method) developed has been focused. These methods are based on nucleic acid detection and quantification of nucleic acid/nucleic acid hybridization (h y b r i d i z a t i ο η ) reaction, and are in principle the same as the conventional methods. 6 312ΧΡ / invention manual (supplement) /94-0S/94 ] 08898

1354792 These methods can be applied to the detection of proteins or sugar chains based on the specific reaction between proteins/proteins, sugar chains/sugar chain chains/proteins. These techniques are used on a glass substrate sheet called a microarray or a D N A wafer, and a high-density arrangement of a large number of DNA fragments or sugar chains is an important feature. For the specific use of the DNA wafer method, for example, a sample obtained by expressing a cell to be examined, such as a fluorescent dye, is hybridized on a planar substrate sheet to bind complementary nucleic acids or RNAs to each other. The resolution detecting device (the method of sweeping the high-speed reading; or detecting the response of the current value according to the electrochemical reaction. Thus, the base S in the sample can be quickly inferred, and the application range of the DNA wafer is not It is only expected to analyze the expression of the amount of expressed genes, and the detection of single nucleotide polymorphism (SNP) as a gene is also expected. As a technique for immobilizing nucleic acids on a substrate, coating on a flat substrate for glass has been developed. a method of immobilizing each nucleic acid using a so-called device (s ρ 〇11 er ), such as poly-L-lysine, amino decane, etc. (曰利特表平1 0 - 5 0 3 8 4 1 Also, a nucleic acid probe (nucleic acid immobilized on a DNA chip) which has recently been used for a DNA wafer has been replaced by a conventional one because it can reduce errors in hybridization with a sample and can be easily synthesized by a machine. Hundreds to thousands of tests For the length and its fragments, oligo DNA (oligo DNA refers to the number of bases of 10 to 100). At this time, the oligo DNA and the glass substrate are combined by a covalent bond (曰利特开2 0 0 1 - 1 0 8 6 8 No. 3) At present, DNA chips are placed on the wafers in the thousands to tens of thousands of 3] 2XP / invention manual (supplement) /94-08/94108S98 or 丨丨J, The amount of the glass flat protein, the gene (DNA S instrument), etc. The gene means tablets and other dot-specific substrates are combined with the cDNA test basic poly 7 7354792

Because it is used for the research purposes of investigating the performance of a large number of genes. In the future, the use of DNA chips is also expected for diagnostic purposes. When using DN A wafers for diagnostic purposes, it is usually pre-determined that the amount of specimens that can be taken is very small. Since the sensitivity of the current DNA wafer is insufficient, the measurement of such a specimen is expected to be impossible. Furthermore, with the current DNA wafers, the low-intensity gene has a very weak fluorescence intensity after hybridization, and these genes have problems that are substantially unresolvable. Therefore, in the case of the current D N A wafer, in the case where the amount of the sample is small or the gene having a small amount of expression, how to increase the fluorescence intensity after hybridization is a problem. In order to solve this problem, how to efficiently react the sample D N A with the probe D N A is critical. As a method of efficiently reacting the sample DNA with the probe DNA, since the natural diffusion by the sample is not sufficient, it is considered to stir the solution to promote efficient reaction between the probe and the sample. As an example of the agitating sample solution, it is disclosed in Japanese Patent Laid-Open Publication No. H02- 2 4 8 0 0 A method of moving the magnetic beads in the sample solution to agitate the sample solution to increase the reaction efficiency with the sample. Further, Japanese Laid-Open Patent Publication No. Hei 2 0 0 3 - 3 3 9 3 7 5 discloses that a sample solution in which beads are mixed is brought into contact with a DNA wafer, and the solution is sealed with a glass cover sheet (c 〇 verg 1 ass). A method of stirring a sample solution by rotating a wafer to cause the beads to fall in the direction of gravity to increase the signal after hybridization. However, the method disclosed in Japanese Laid-Open Patent Publication No. 2000-240-108 That is, in general, a flat-shaped D N A wafer is used, and a conventional glass cover is used. 8 312XP/invention specification (supplement)/94-08/94108898 1354792

When the sheet seals the sample solution, the gap between the cover glass and the DNA wafer is about 10 // m. Therefore, when the larger particles are mixed, the particles are sandwiched between the DNA wafer and the cover glass, and the particles cannot move, so that the problem of the effect cannot occur. Further, in the case of the microparticles having a size of several m, even if the particles are to be moved by gravity or the like, the microparticles do not sufficiently move in the sample solution due to the resistance of the solution, and the effect of the stirring cannot be sufficiently exerted. Further, even if the fine particles are moved by gravity, it is estimated that the contact of the fine particles with the surface of the carrier to which the DNA probe is immobilized is a factor that cannot be obtained. Further, a method in which the distance between the cover glass and the DNA wafer is increased by the 0-ring or the like, the fine particles for stirring are enlarged, and the fine particles in the reaction liquid are moved by gravity or magnetic force to stir the solution. However, since both the cover glass and the DNA wafer for sealing are flat, the fine particles are also moved in the portion where the DNA probe is fixed. Therefore, the microparticles may damage the portion where the probe D N A is immobilized, because the damage may cause an obstacle in data analysis, or the probe may be peeled off due to the microparticle damage and the probe fixing surface, so that the signal strength is weakened. SUMMARY OF THE INVENTION The present invention is a solution agitation method in which a selective binding substance immobilized on a surface of a carrier is contacted with a solution containing a substance to be tested which reacts with the selective binding substance, and the solution is stirred; A method of mixing fine particles or bubbles in a solution containing a test substance to move the fine particles or bubbles under contact with a fixed surface of the selective binding substance to stir the solution. [Embodiment] 9 312XP/Invention Manual (Supplement)/94-08/94108898 1354792 Hereinafter, the stirring method of the present invention will be described. In the first method of stirring a solution according to the present invention, a selective binding substance immobilized on a surface of a carrier is contacted with a solution containing a substance to be tested which reacts with the selective binding substance, and the solution is stirred; A fine particle or a bubble is mixed in the solution of the test substance so that the fine particle or the bubble does not contact the fixed surface of the selected binding substance to move the stirring solution. In the solution agitation method of the first aspect of the invention, it is necessary to stir the solution by mixing fine particles or bubbles in the solution containing the test substance and moving the fine particles or bubbles. Further, in the first method of stirring a solution according to the present invention, the fine particles or the bubbles are moved while being in contact with the fixed surface of the selective binding substance to stir the solution. By restricting the movement of the particles or bubbles, it is possible to prevent the particles from being damaged by the particles or bubbles due to the contact of the particles with the fixed surface of the probe.

It is preferred to use a carrier of a structure in which fine particles or bubbles are not in contact with the fixed surface of the selected binding substance. It is preferable to provide the uneven portion on the carrier and to fix the selective binding substance on the convex portion. Further, it is preferable to use a container for holding a solution in which microparticles or bubbles are not in contact with the fixing surface of the selective binding substance. Furthermore, in the second method of stirring a solution according to the present invention, the selective binding substance immobilized on the convex portion of the carrier is brought into contact with a solution containing the test substance which reacts with the selective binding substance, and the solution is contacted with the solution. The agitator is mixed; the microparticles or bubbles are mixed in a solution containing the test substance, and the microparticles or bubbles are moved to stir the solution. 10 3 ] 2XP/Invention Manual (Supplement)/94-08/94108898 1354792 In the first solution stirring method of the present invention and the second solution stirring method, bubbles or fine particles are used. The first solution stirring method and the second solution stirring method according to the first aspect of the present invention are preferably microparticles, as compared with the case where the size and the material are selected and the specific gravity can be easily controlled.

In the solution stirring method of the present invention, the size of the fine particles (the maximum diameter of the fine particles) is preferably 10 / z m or more. If the size of the microparticles is less than 10 // m, the effect of stirring by the microparticles is hardly obtained. The reason is that if the size of the fine particles is less than 10 # m, even if an external force (magnetic field, gravity, or vibration) is applied, the particles are hardly moved due to the P and the force of the solution. It is particularly preferable that the fine particles are more than 2 0 /z m or more. In the solution stirring method of the present invention, fine particles of any shape can be used. Particularly preferred is that the shape of the microparticles is spherical, that is, beads. When the microparticles are beads and are rotated by themselves, they can be smoothly moved without stopping in the reaction liquid, and as a result, the sample solution can be satisfactorily stirred, which is preferable. As the form of the fine particles, spherical fine particles (beads) having a diameter of 2 0 // m - 3 0 0 m can be preferably used. If the diameter of the bead is within this range, the gravity of the bead itself can easily move the bead in the liquid by gravity or acceleration even if there is resistance of the reaction liquid, and the liquid can be sufficiently stirred. Therefore, good results can be obtained. In the solution stirring method of the present invention, the material of the fine particles is not particularly limited. The material of the microparticles may be metal, glass, ceramic, or polymer (polystyrene, polypropylene, nylon, etc.). Among these, if the material is larger than the water (glass, quartz, cerium oxide ceramic) beads, due to gravity or 11 3] 2XP / invention manual (supplement) /94-08/94108898 1354792 It is preferable that the acceleration generated by the vibration or the like can be easily moved in the liquid. Further, magnetic beads can also be used. In particular, since the magnetic beads composed of the zirconia ceramic have a large specific gravity, the beads can be easily moved by the acceleration generated by gravity or vibration, so that it is an optimum user. Further, since glass, quartz, and zirconia ceramics have less elution of the bead component in the sample solution, it is preferable.

The beads composed of cerium oxide ceramics (cerium oxide stabilized cerium oxide) have a density of 6 g/cm 3 , which is larger than 2.2 g/cm 3 of quartz glass, so that the stirring effect can be exerted. When the container is sealed, the movement of the solution is less, and the beads are less likely to move up, so it is easier to set and is particularly good. In the solution stirring method of the present invention, it is preferred to move the fine particles to agitate the solution. In the solution stirring method of the present invention, it is more preferable to move the fine particles by any one of gravity, magnetic force, vibration of the carrier, or a combination thereof. Among them, the method of moving the carrier along the vertical plane and moving the beads by gravity is preferable because it can be easily carried out and a sufficient effect can be obtained. The rotation speed at this time is preferably 0.1 rpm to 30 rpm. If the rotational speed exceeds 3 0 r p m, if the microparticles do not move completely in one direction, there will be a case where the opposite side of gravity is applied to the microparticles. That is, the distance at which the microparticles move back and forth in the sample solution is reduced, so that the effect of the agitation cannot be sufficiently exerted. Further, when the rotational speed is slower than 0.1 r p m , the total time of movement of the fine particles in the liquid becomes short, and as a result, the stirring time of the sample solution becomes short, so that a sufficient effect cannot be obtained. In view of the above, the rotation speed is preferably in the range of 0.5 rpm to 5 rpni. It is also a preferred method to move the particles in the solution by vibrating the carrier to the left and right and applying acceleration. 12 312XP/Invention Manual (Supplement)/94-08/94丨08898 1354792 In the solution stirring method of the present invention, it is preferred to use a container for holding the solution. Further, in the solution stirring method of the present invention, it is more preferred to move the solution by stirring the microparticles, and the minimum width of the microparticles is larger than the shortest distance between the fixing surface of the selective binding substance and the container holding the solution. In the solution stirring method of the present invention, it is preferable that the maximum width of the fine particles is 10 μm or more and the height difference between the upper surface of the convex portion and the concave portion is equal to or less. In the solution stirring method of the present invention, it is preferred that the fine particles are moved to agitate the solution, and the uneven portion is provided on the carrier, and the bonding substance is fixed on the convex portion to move the fine particles in the concave portion. In the solution stirring method of the present invention, it is preferable that the flat portion and the uneven portion are provided on the carrier, and the selective bonding substance is fixed on the plurality of convex portions, and the heights of the convex portions are substantially the same, and the flat portions and the convex portions are The height difference above is 50//m or less. The preferred shape of the carrier to which the selective binding substance is immobilized will be described.

The carrier to which the binding substance is selected for use in the stirring method of the present invention is preferably provided with a concavo-convex portion and a selective binding substance to be fixed on the convex portion. By adopting such a structure, since the non-specifically adsorbed sample is not detected at the time of detection, there is less noise, and as a result, a better S/N result can be obtained. The specific reasons why noise is reduced are as follows. That is, the carrier that is selected to be bonded to the convex portion is scanned by a device called a scanner, and the focus of the laser light is concentrated on the convex portion of the concave and convex portion, so that the laser light is not focused on the concave portion, but It is not easy to detect the effect of the non-specific amount of fluorescence (missing 13 312XP/invention specification (supplement)/94-08/94108898 1354792) that is not specifically adsorbed on the concave portion. It is preferable that the height of the convex portion of the uneven portion is substantially the same as the height of the upper surface of each convex portion. Here, the term "the height is substantially the same as j" means that the selected binding substance is fixed to the surface of the convex portion having a certain difference in height, and is reacted with the fluorescently labeled subject, and then scanned by the scanner. The difference in signal level is not at a problematic height. Specifically, the so-called "height is about the same" means that the height difference is less than 5 Ο μ m. It is more preferable that the height difference is less than 30 /zm or less, and the height is preferably the same. Further, the same height as referred to in the present application # includes an error that occurs in production or the like. If the difference between the height above the highest convex portion and the height of the lowest convex portion is greater than 50. #m, the laser light above the convex portion whose height is deviated will not be easily focused, and the selective combination with the convex portion is fixed. The intensity of the signal emitted by the sample in which the substance reacts will be weak. Further, it is preferable that the upper surface of the convex portion is substantially flat. Here, the term "the upper surface of the convex portion is substantially flat" means that the unevenness does not exceed 20 /z m or more.

Further, it is preferred that the carrier used in the stirring method of the present invention is provided with a flat portion. The height of the upper surface of the convex portion of the uneven portion is preferably substantially the same. That is, the height difference between the flat portion and the upper surface of the convex portion is preferably 5 0 /z m or less. If the height difference between the height of the convex portion and the flat portion is 50 μm or more, the detectable fluorescence intensity is weak. More preferably, it is 3 0 // πι or less, and it is preferable that the height of the flat portion is the same as the height of the convex portion. Specific examples of the carrier used in the stirring method of the present invention are shown in Figs. 3 and 4. There is a flat portion indicated by 1 1 around the uneven portion, and is 14

312XP/Invention Manual (Supplement)/94-08/94] 0889S 1354792 12 The selective convex substance (for example, nucleic acid) is fixed to the convex portion of the uneven portion. With this flat portion, it is easy to focus on the top of the convex portion. That is, when the focus of the light is focused, the focus of the laser light is adjusted in advance as shown in FIG. 5 to make the surface used in the stirring method of the present invention, and the focus of the laser light can be easily made in the solution stirring method of the present invention. Selection of the carrier of the substance: the binding substance necessary for the binding of the substance as the data, the exclusion of only the pseudo-selection is selected in the solution stirring method of the present invention, and the area above the surface of the convex portion of the carrier of the substance is The area of the various parts can be the same, so the area of the upper part of the convex part is roughly

The area of the upper surface is divided by the minimum of the carrier in the solution stirring method of the present invention, and the area of the upper surface of the convex portion and the amount of the substance may be less than the μ m 2 or more which is easy to handle. In the solution stirring method of the present invention, the height of the convex portion is 10 0 m or more, and the focus scanner of the excitation light of the scanner is performed on the surface of the carrier at 50 0 in or more and 300 μm. As a result of the excitation, the carrier body is mostly placed against the height of the jig and the abutment surface of the jig. The flat portion of the carrier abuts against the convex portion of the holder that focuses on the carrier. The term "fixed plurality of convex portions fixed by the combination of binding properties" means a fixed portion of a specific substance to which a substance (for example, a nucleic acid) is immobilized. Preferably, the fixed selective binding product is substantially the same. The selection of the binding substance by the selection of the convex portion facilitates subsequent analysis. Here, "the same" means that the maximum product in the convex portion has a value of 1.2 or less. The selective binding substance is not particularly limited, and from the viewpoint of bonding, it is preferably 1 m 2 or less '1 0 , which is preferably 0 or less of the concave portion of the carrier to be used. It is particularly preferable based on the following. The height of the convex part is 15 312XP / invention manual (supplement) /94-0S/94 ] 0S898

1354792 is lower than this, and the non-specifically adsorbed sample outside the focus will also be detected, resulting in S / N deterioration. Further, if the height of the convex portion is equal to or greater than the shoulder #m, the problem that the convex portion is broken or broken is likely to occur. In the first solution stirring method of the present invention, there are restrictions on the fine particles or the gas moving region. The shape of the container for holding the carrier and the solution for realizing this point will be described with reference to Fig. 1 as an example. In Fig. 1, 1 indicates a probe D N A (selective binding substance). Further, 2 particles (in this case, beads), and 3 are carriers to which the probe D N A is immobilized. This 2, 3 series contacts a solution containing the standard D N A (test substance). Further, 4 is a glass slide, a glass cover sheet, or a container made of a material such as metal or plastic, and a solution containing the target DNA is held between the containers. In the example of Fig. 1, the probe DNA is immobilized on the carrier of the carrier, the shortest distance from the convex portion of the carrier (the surface on which the binding substance is selected) and the container of the holding liquid is the diameter of the microparticles, forming no particles. The state in which the surface of the probe DNA is fixed is in contact with the surface of the probe DNA. In the case where the fine particles are, for example, elliptical, if the shortest distance from the upper surface of the convex portion is less than the minimum width of the fine particles, the contact of the probe with the fine particles or the bubbles can be prevented. As a method for specifically realizing the situation of FIG. 1, a solution containing a sample D Ν ( (sample solution) is dropped onto a carrier having irregularities, and fine particles are placed in the liquid in such a manner that fine particles are attached to the convex portion. Thereafter, the glass cover sheet corresponding to the container is covered, and the periphery thereof is sealed with an adhesive or an adhesive or the like so as not to overflow or evaporate the sample solution. For example, between the surface of the cover glass and the top of the convex portion, there may be several / / m ~ tens of / / 312XP / invention instructions (supplement) / 94-0S / 94108S98 sample, 500 bubble is micro-etc. The protection and the loading portion are so dissolved that the micro-injury and the shape of the container are not made to fit the space of the sample solution by the D1 band, I il 匕, m path 16 1354792 degrees. If the size of the microparticles is larger than the surface between the cover glass and the upper surface of the convex portion, the fine particles do not damage the upper surface of the convex portion. By using the carrier of such a shape, the carrier is rotated in a vertical plane, and the fine particles are moved only in the concave portion of the uneven portion, and the sample solution can be stirred without touching the convex portion. In order to make sure that there is a space for injecting the sample solution between the upper surface of the convex portion and the container, it is preferable to prepare, for example, a plate having a corner of the plate surface higher than other surfaces by 5/zm to 100//m; or a central portion; A plate of 5 #m to 1 0 0 #m is recessed; and the center portion of the plate is fitted so as to compete with the uneven portion of the carrier to which the conjugated material is selected. An example of this board is shown in Figure 1 of Figure 1. To make such a container, for example, a film or an adhesive tape may be applied to the side of the glass to which the glass is treated with hydrofluoric acid; or a plate of the shape of FIG. 1 may be formed by injection molding or the like; or may be printed by screen printing. The corners of the board are printed with ridges and the like. In the solution agitation method of the present invention, it is preferred to use a container which holds a solution in which fine particles or bubbles are not in contact with the fixing surface of the selective binding substance.

The carrier in Fig. 1 has a concavo-convex shape. The same effect can be obtained by providing the concavo-convex shape in the container holding the solution. A specific example thereof is shown in Fig. 2 . In this case, the probe DNA is placed under the convex portion of the container. In this case, the distance between the surface of the probe DNA and the convex portion of the container may be less than the minimum width of the microparticles. As another specific example, the structure of both the carrier and the container can be used as the uneven structure. The carrier provided with the uneven portion or the container provided with the uneven portion as described above is used to stir the sample solution containing the target DNA by the microparticles. 17 312XP/Invention Manual (Supplement)/94-08/94〗 OS898

1354792 Mixing, the following effects can be exerted, and as a result, the intensity of the fluorescence intensity after hybridization is stronger. That is, in the case where the glass cover sheet is hybridized by a general flat DNA wafer, the gap between the glass cover sheet and the DNA wafer is as high as about "m. Even if a larger particle size is mixed, the microparticles are trapped between the DNA wafer and the cover glass, so that the microparticles cannot move, and the effect of mixing the microparticles is lost. On the other hand, in order to avoid this, the mixing is not sandwiched between the glass cover sheet and the DNA wafer, and even if the microparticles are moved by the acceleration of gravity or vibration, since the microparticles are small, It will withstand a large solution resistance so that the particles cannot move sufficiently in the sample solution. Therefore, the problem that the stirring effect of the child cannot be fully exerted. Moreover, if the distance between the cover glass and the carrier of the 0-ring is increased, and the fine particles for stirring are sufficiently stirred, the surface of the wafer is damaged by the microparticles, and the microparticles are impinged on the probe. The probe is detached from the surface, so there is a problem that the fluorescence intensity after the intersection is not strong enough. According to a preferred embodiment of the present invention, if a container provided with a concave-convex portion or a concave-convex portion is used, as shown in FIG. 1 or FIG. 2, the size of the sub-portion can be at least increased from the concave portion to the convex portion of the concave-convex portion. In the height of the portion, the sample containing the uneven portion or the sample having the uneven portion is used to stir the sample solution containing the target DNA, and the larger particles can sufficiently stir the sample solution, and the sample is not stirred. The fixed surface of the probe DNA can obtain a better effect. In the solution stirring method of the present invention, the material of the suitable container is 312XP/invention specification (supplement)/94-08/94108S98, which is carried out for 10 times in the funeral problem, t β m particles, causing the particles to make the glass large Or push the particles loaded with impurities. Factor, by injury, no 18

1354792 Specially limited. In the present invention, as a material of a suitable container, it may be, for example, glass or plastic. When the shape of the container is a flat plate, a glass plate such as a glass cover sheet or a glass slide is preferably used. On the other hand, when the shape is an uneven shape, the production surface for injection molding can be used. It is preferably a plastic material such as polymethyl methacrylate or polycarbonate. The material of the carrier used in the present invention is not particularly limited. In this context, the more suitable carrier material is glass or various polymers (polyphenylethylene phthalate, polycarbonate). In order to fix the selective binding substance, the material of the carrier is glassy, and a functional group can be formed on the surface by decane coupling treatment, whereby the selective binding substance such as D N A can be fixed to the substrate on the carrier. For example, an amine group is formed on the surface of the glass by using an aminoalkyl decane or the like, and the DNA is combined, whereby the positive charge of the amine group and the negative charge of the DNA can be fixed by static. In the present invention, in particular, when the surface for fixing the selective binding substance is a solid having a structural unit represented by the following general formula (1), the signal after hybridization becomes large. Therefore, it is better. R1 CH, -C- C=0

I

X t R2 (R1, R2, and R3 in the formula (1) represent an alkyl group, an aryl group or a hydrogen atom.) As a polymer having a structural unit represented by the formula Π), 312XP/invention specification (supplement) /94-08/94] 08898 For example, a container-like square rubber may be invented as a field power carrier (1) X = 0, NR3, CH2 using 19 1354792 single polymer or copolymer. The polymer uses at least one type of monomer as a raw material, and the single system exists in the form of a double bond which can participate in polymerization, a functional group which can participate in polycondensation, and a derivative of a ketone or a carboxylic acid or the like. Shape. Further, the above polymer is more preferably a structure having the formula (1). When the polymer containing the structural unit represented by the formula (1) is a copolymer, it is preferred that the structural unit represented by the formula (1) is more than 10% by weight of all the monomer units. When the content Φ of the structural unit represented by the general formula (1) is 10% or more, a large amount of carboxyl groups can be formed on the surface by a step described later, and a large amount of probe nucleic acid can be immobilized, resulting in s / N can be more mentioning. In the present invention, the term "polymer" means a number average degree of polymerization of 50 or more. The preferred range of the average degree of polymerization is from 1 0 0 to 1 0 0 0 0. Especially, it is especially preferable that it is 200 or more and 500 or less. Further, the number average degree of polymerization can be easily determined by measuring the molecular weight of the polymer by a predetermined method using G P C (gel permeation chromatography).

In the formula (1), R1 and R2 represent an alkyl group, an aryl group or a hydrogen atom, and may be the same or different. The above alkyl group may be linear or branched, and preferably has 20 carbon atoms. The above aryl group is preferably 6 to 18 carbons, more preferably 6 to 12 carbon atoms. The functional group X may be arbitrarily selected from 0, NR3, or CH2. R3 is a functional group defined by the same as the above R1 and R2. In the present invention, the polymer for the surface of the support to which the binding substance is selected is preferably a polymer containing a functional group. The polymer having a functional group is preferably, for example, poly(methyl methacrylate) SI ( Ρ Μ Μ A ), polyfluorenyl 20 312XP / invention specification (supplement) / 94-0S/94108898

1354792 Ethyl acrylate (PE Μ A) or polymethacrylate (P A Μ A ) of polypropyl methacrylate. Among them, polymethyl methacrylate is particularly preferable, and polyvinyl acetate, polycyclohexyl methacrylate or polyphenyl acrylate can also be used. Further, a copolymer of a structure in which the composition of the polymer is combined may be used, or a structural composition in which a constituent of the polymer of one or more of the other polymers is added to the composition of the polymer. The other polymer described above is polystyrene. When the polymer is a copolymer, the ratio of the ratio of each component is preferably 10 mol% or more of a carboxyl group-containing monomer (for example, an alkyl methacrylate). According to this, since a large amount of probe nucleic acid can be immobilized by forming a polycarboxy group on the surface, the result can be further improved. Among the structural units of the polymer, a more preferable ratio of the monomer is 50% or more. In order to immobilize the selective binding substance on a carrier comprising a polymer having at least one structure represented by the formula (1), it is preferred to form a carboxyl group on the surface of the carrier. As means for forming the surface of the carrier, it may be treated by acid, alkali or the like, sonication in warm water, exposure of the carrier to oxygen gas, argon plasma, radiation, etc., from damage to the carrier. From the viewpoint of being less and easy to perform, it is preferred that the carrier is impregnated with a base or an acid to form a carboxyl group on the surface. Specifically, the carrier may be immersed in an aqueous solution of sodium hydroxide or sulfuric acid (compared to a concentration of 1 N to 2 0 N ), preferably at a temperature of 30 ° C to 80 ° C, for 1 hour to 1 0 0 hours. As the polymer, it is also possible to use a thermoplastic 312XP/invention specification (supplement)/94-08/94108898 acid anhydride having an acid anhydride unit. The re-copolymerization of the methyl elemental element, for example, the amount of S/N molar units previously formed into a super-linear carboxy group, as a good example of maintaining the polymerization 21 1354792. This thermoplastic copolymer preferably has (i) an anhydride unit. The term "(i) anhydride unit" is a unit present in the skeleton or terminal of the main side chain of (A) thermoplastic copolymer. The structure of the (i) acid anhydride unit is not particularly limited to fil, and examples thereof include a (meth)acrylic anhydride unit, a pentane unit, a maleic anhydride unit, an itaconic anhydride unit, a pyroic acid group, and an aconite unit. The maleic anhydride unit or the glutaric acid group is preferred, and the glutaric acid group chain or the anhydride anhydride monoanhydride unit anhydride unit represented by the following formula (2) is more preferred.

(2) (In the above formula, R4 and R5 represent the same or different hydrogen atoms or carbon number 1·alkyl groups). The structure of the thermoplastic copolymer is not particularly limited as long as it contains the (i) acid anhydride unit, and preferably has (i i ) and a carboxylic acid unit represented by the following general formula (3). -5 can, not full

Ping 6 \ CH2—C-coo^/ (3) (wherein R6 represents hydrogen or an alkyl group having 1 to 5 carbon atoms). Here, "(i saturated carboxylic acid unit) means that the unsaturated carboxylic acid monomer is united by copolymerization, and the unsaturated carboxylic acid monomer used at this time is not particularly limited. i) 312XP/ BRIEF DESCRIPTION OF THE INVENTION (Supplement) /94-08/94108898 22 1354792 Any unsaturated carboxylic acid monomer copolymerized with other vinyl compounds can be used. The preferred unsaturated carboxylic acid monomer is exemplified by the following formula (4) ch2 di c (4)

COOH. (wherein, R6 represents hydrogen or an alkyl group having 1 to 5 carbon atoms), a hydrolyzate of maleic acid or maleic anhydride, and the like, in terms of excellent thermal stability, Acrylic acid and methacrylic acid Φ are preferred, and methacrylic acid is more preferred. These may be used in one type or at least two types. (A) The thermoplastic copolymer is not particularly limited as long as it contains the (i) acid anhydride unit, and preferably has the following formula (5)

(wherein R7 represents hydrogen or an alkyl group having 1 to 5 carbon atoms, and R8 represents an aliphatic or alicyclic hydrocarbon group having 1 to 6 carbon atoms or 1 or more hydroxyl groups or 6 or less carbon atoms substituted by halogen; An aliphatic or alicyclic hydrocarbon group) is represented by (iii) an unsaturated carboxylic acid alkyl ester unit. The "(iii) unsaturated carboxylic acid alkyl ester unit" referred to herein is a unit obtained by copolymerization of an unsaturated carboxylic acid alkyl ester monomer, and here, as an unsaturated carboxylic acid alkyl ester single. The body is not particularly limited, and a preferred example thereof is represented by the following formula (6). 23 312XP/Invention Manual (Supplement)/94-08/94 ] 08898 1354792 R7 cn2—c (6) COOR8 On the surface of the carrier, as long as there is a carboxyl group or an acid anhydride, a selective binding substance having an amine group or a hydroxyl group can be used. The surface of the carrier is fixed by a covalent bond. In the case where a carboxyl group is present on the surface of the carrier, in order to promote such a bonding reaction, dicyclohexylcarbodiimide or N-ethyl-5-phenyliso is used. Various condensing agents such as oxazoline-3'-sulfate. Among them, especially 1-ethyl-3-(3-difluorenylaminopropyl)carbodiimide (EDC), because of its low toxicity and relatively easy to remove from the reaction system, The condensation reaction of the binding substance with the carboxyl group on the surface of the carrier is one of the most effective condensing agents. These condensing agents such as EDC may be used in combination with a solution for selecting a binding substance, or a carrier having a carboxyl group formed on the surface may be first impregnated into a solution of EDC, and the carboxyl group on the surface may be activated. The condensing agent can be used in combination with a solution for selecting a binding substance to increase the reaction yield, and a plurality of selected binding substances can be immobilized on the carrier, which is suitable.

When such a condensing agent is used to react the carboxyl group on the surface of the carrier with the amine group of the selective binding substance, the selective binding substance is immobilized on the surface of the carrier by a guanamine bond; the carboxyl group on the surface of the carrier and the selective binding substance are used. In the case of a hydroxyl group reaction, the selective binding substance is immobilized on the surface of the carrier by ester bonding. The temperature at which the sample containing the selective binding substance acts on the carrier is preferably from 0 ° C to 95 ° C, more preferably from 15 ° C to 65 ° C. Processing time is usually » 5 minutes to 2 4 hours, preferably more than 1 hour. On the other hand, in the case of a polymer having an acid anhydride on the surface, it can be added to 24 312XP / invention specification (supplement) /94-08/94108898 1354792

The above-mentioned condensing agent can be covalently bonded without adding an amine group, and the selected binding substance can be fixed by selective adsorption and non-specific adsorption of the sample, and the selective binding property can be selected. A carrier with high efficiency of hybridization of the space of matter. In the case where the carrier is prepared by the general formula (η or the general formula Φ, and the glass, by the injection molding method or the hot embossing (h is mass-produced with a fine uneven method, since it is easy to mass-produce, it is used in the present invention. Preferably, the selected binding sample is adsorbed on the surface of the polymer, and the material is immobilized by a covalent bond, and the carrier having a higher degree of spatial freedom with the vitrified substance is obtained by the above method. Selecting the binding substance to fix and then applying the alkali treatment, surfactant treatment, etc. The binding substance is selected to fix the carrier to the carrier, for example, the binding substance is fixed to the polymer. The surface can be strongly and highly densely covalently. It is inferred that the solidification degree is higher than that of the glass, so that the polymer of the structural unit represented by the sample (2) can be obtained. By comparison of the isophase, the carrier of the shape can be more simply formed by the ot emboss method or the like. In particular, the carrier which is preferably formed by injection can be fixed by the substance by the above method. The non-specificity of the solid and high-density comparison of the selected binding glass is inferred by the fixed selection knot, so that the hybridization efficiency binding substance with the sample can be obtained, and the treatment can be selected. For example, By heat treatment, it is also possible to immobilize the selected binding substance, usually by hybridizing the sample of the fluorescent label with the substance, which is called scanning 25 312XP/invention specification (supplement)/94-08/9410S8卯

The device of 1354792 reads the fluorescent light. The scanner concentrates the laser light by focusing it with a laser that belongs to the excitation light. However, in the case where the fluorescent light is generated from the surface of the carrier, the light emission becomes noise and the detection accuracy is lowered. In this case, in order to reduce the fluorescence emitted from the carrier itself, it is preferred that the polymer of the structural unit of the formula (1) or (2) appears black, or contains a laser that does not emit light. The substance is made black by using such a carrier, and the fluorescence from the carrier itself can be reduced at the time of detection. The black carrier is small in noise, and as a result, it can be a carrier of S/N ratio # fixed binding substance. Here, the term "black is a carrier" means that the spectral reflectance of the black portion of the carrier has a specific spectral pattern (specific peaks, etc.) in the visible light range (waves of 400 nm to 800 nm), which is the same low value. The light transmittance of the black portion of the body also has no specific spectral pattern, which is a low value. In the present invention, the carrier preferably has a spectral reflectance of 7% or less in the range of visible light (waves of 400 nm to 800 nm) and a light transmittance of 2% or less at the same wavelength. Here, the spectral reflectance is suitable for the spectral reflectance in the case of the illumination of the condition C of the J I S Z 8 7 2 2 condition, and the case where the body obtains the specular reflected light. In the present invention, the method of making the carrier black may be achieved by the inclusion of a black substance in the body, and the preferred black substance may be exemplified by carbon black 'graphite, black, aniline black 'R u, Μ π, N Black matter of i, C r , F e , C u oxide, carbide of Si, T i, T a, Z r and C r . Among these black substances, it is preferable to contain carbon black, graphite, and titanium black. 3] 2XP / invention manual (supplement) / 94-08/94108898 The object is protected against color. It is good to use it. It is not used and the length is the same. It means that it is self-loading, such as: 〇 and 色 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 These black substances may be contained singly or in combination of at least two kinds.

In the present invention, as the shape of the carrier, a polymer having at least one structural unit represented by the general formula (1) is provided on a support composed of a material which is not easily thermally deformed such as glass or metal. It is preferable that the composition is combined with the fixing layer of the substance to prevent the shape change of the carrier due to heat or external force. An example of this concept is shown in Figure 6. As the support layer, a metal Φ such as polypropylene, glass, iron, chromium, nickel, titanium or stainless steel is preferred. Further, in order to improve the adhesion between the support layer and the selective binding substance fixing layer, it is preferred to subject the surface of the support layer with a plasma treatment of argon gas, oxygen gas or nitrogen gas or a treatment with a decane coupling agent. Examples of such a decane coupling agent include 3-aminopropyltriethoxydecane, 3-aminopropyltrimethoxyxanthine, and 3-aminopropyldiethoxysulfonyl sulphate. 3-(2-aminoethylaminopropyl)trimethoxy decane, 3-(2-aminoethylaminopropyl)dimethoxymethylnonane, 3-thiolpropyltrimethoxy Decane, dimethoxy-3-thiolpropyl fluorenyl sulphate and the like. As a means for providing a fixing layer for selecting a binding substance on the support layer, a known means for dissolving the polymer in an organic solvent and performing spin coating or dipping can be used. More simply, an adhesive can be used to bond to the support layer. In the present invention, the term "selective binding substance" means a substance which can be selectively and directly or indirectly bonded to a test substance, and examples thereof include nucleic acid, protein, saccharide and other antigenicity. Compound. As a "selective binding substance", a particularly good one is a nucleic acid. The nucleic acid may be DNA or RNA or PNA. a strand having a specific base sequence 27 312XP / invention specification (supplement) /94-08/9410S898 1354792 acid, and a strand having a base sequence complementary to the base sequence or a part thereof Since the nucleic acid is selectively hybridized and combined, it corresponds to the so-called "selective binding substance" in the present invention. Further, examples of the protein include an antibody and a F a b fragment or F ( a b ′ ) 2 .

An antigen-binding fragment of an antibody such as a fragment, and various antigens. The antibody and its antigen-binding fragment selectively bind to the corresponding antigen, and the antigen is selectively bound to the corresponding antibody, so it is equivalent to "selective binding substance". As the saccharide, a polysaccharide is preferred, and various antigens can be exemplified. Further, it is also possible to fix a substance other than protein or saccharide having antigenicity. The selective binding substance used in the present invention may be a commercially available one or a biological cell or the like. The selective binding substance used in the present invention is preferably a nucleic acid, particularly a nucleic acid having a length of 10 bases to 10 0 bases, which is called an oligonucleic acid, which can be easily synthesized by using a synthesizer. Further, since the amino group at the terminal of the nucleic acid is easily modified, it is easy to be immobilized on the surface of the carrier, and is preferable. Further, if the stability of hybridization is low from the viewpoint of less than 20 bases, it is more preferable to use 20 to 100 bases. In order to maintain the stability of hybridization, the range of 40 to 100 bases is particularly preferable. In the solution stirring method of the present invention, the nucleic acid to be measured as the test substance may, for example, be a gene such as a pathogen or a virus, a gene for genetic diseases, a part thereof, and various biological components having antigenicity. The antibodies to pathogens, viruses, and the like are not limited thereto. In the solution stirring method of the present invention, as a solution containing such a test substance, 28 312XP/invention specification (supplement)/94-08/94108898 1354792 solution is extracted. (t-nuclear Φ substance storage body pair

For example, blood, serum, plasma 'urine' feces, spinal cord, saliva, body fluids such as various tissue fluids, various foods and the like, and the like, are not limited thereto. Wait. The nucleic acid to be a test substance can be identified by a nucleic acid obtained by a usual method from blood or cells, or can be amplified by a nucleic acid amplification method such as PCR using the nucleic acid as a template emplate. With the acid as a template, the measurement sensitivity can be greatly improved by the field magnified by the nucleic acid amplification method such as PCR. When a nucleic acid-amplified product is used as a sample, the amplified nucleic acid can be labeled by amplification under a nucleotide triphosphate identified by a fluorescent substance or the like. Further, when the substance to be detected is an antigen or an antibody, the antigen or antibody of the test substance can be directly identified by a usual method. Alternatively, after binding the antigen or anti-substance of the test substance to the selective binding substance, the vector is washed, and the antigen or anti-reaction is reacted with the antibody or antigen which is identified as an antigen-antibody reaction, and is bound to the label of the carrier. Determination. In the solution stirring method of the present invention, it is preferred to react the selected binding substance with the test substance. In the solution stirring method of the present invention, the step of allowing the immobilized substance to react with the test substance can be carried out by the same method as conventionally known. The reaction temperature time can be appropriately selected depending on the chain length of the nucleic acid to be hybridized, the type of antibody involved in the immune reaction, and/or the type of the antibody, and in the case of hybridization of nucleic acid, it is usually at 35 ° C to 70 ° C. The degree is from 1 minute to more than ten hours, and in the case of no reaction, it is usually carried out at a temperature of from room temperature to 40 ° C for 1 minute to several hours. 312XP/Invention Manual (supplement)/94-08/94 】08S98 29

1354792 In the solution stirring method of the present invention, it is understood that not only the signal but also the advantages described later are obtained. That is, with the conventional method of intersection, the fluorescence intensity after hybridization is weak, and the distribution of the fluorescence intensity in the fixed spot becomes a problem hindering the data analysis after the donut. However, in the present mixing method, not only the fluorescence intensity is greatly improved, but also the donut-like fluorescence intensity distribution in the focus can be reduced. (Examples) The present invention will be further described in detail with reference to the following examples. The invention is not limited to the following examples. (Example 1) (Production of DNA-immobilized carrier) An injection was produced by a LIGA (Lithographie G? Abformung) process by a known method, and a body having a shape described later was obtained by an injection molding method. Further, the average molecular weight PM Μ A of PMMA used in this example contained carbon black in a proportion of 1% by weight (the Mitsubishi Chemical # carrier was black. The spectral reflectance of the black carrier was measured, and as a result, the spectral reflectance was in visible light. The domain (the wavelength is 5% or less at any wavelength, and is 0.5% or less in the same range. The spectral reflectance and the spectral transmittance are the same as those without specific spectral patterns (peaks, etc.). Increasing the hybrid DNA chip by a device suitable for the JISZ 8 7 2 2 primary light receiving optical system (M 乐 - 312XP / invention manual (supplement) / 94-08/94108898) The dosage of the hetero-probe DNA will lead to the stirring of the solution of the invention and the advantages of the above-mentioned ones. For the description, the amount of the PMM A made by the ilvanoformung mold is 50,000, in the system, # 3 0 5 0 B ), rate and spectral transmission through the wavelength range of 400nm ~ 800nm) The transmittance in the visible light region is flat. Also, the illumination of the piece C, 2 0 0 2), for the self-loading 30

1354792 The measurement of the spectral reflectance when the body obtains specular reflected light. The shape of the carrier was 76 mm in length, 26 mm in width, and 1 mm in thickness, and the surface other than the central portion of the body was flat. In the center of the carrier, there is a concave portion having a diameter of 10 mm and a depth of 0.2 mm, and the concave portion has 64 (8 x 8) convex portions having a diameter of 0.2 mm and a height of 0.2 mm. The difference in height between the height of the convex portion on the concave portion (the average value of the heights of the convex portions at 64) was measured and found to be 3 // m or less. Further, the difference in height above the convex portion (the difference between the height of the highest convex portion and the lowest Φ surface), or even the difference between the average height of the convex portion and the height of the plane, is measured as 3 / respectively. /m below. The pitch of the convex portion of the convex portion (the distance from the center of the convex portion to the adjacent convex portion) is 0 · 6 m m. The above PMMA carrier was immersed in 10N sodium hydroxide at 65 ° C for 12 hours. This was washed successively with pure water and 0.1 N of H C 1 pure water to form a carboxyl group on the surface of the carrier. (Identification of probe D Ν 合成) Synthetic SEQ ID NO: 1 (60 base, 5' end is aminated) white The 5' end of the DNA of SEQ ID NO: 1 is aminated. This D N A was dissolved in water at a concentration of 0.3 π m ο 1 / a L to prepare a stock solution. When immersed in the carrier, 8 g of NaCl 2.9 g of Na 2 HP 〇 4 · 1 2 Hz 〇 0. 2 g of KC 1 K Η 2 P 0 4 was dissolved in pure water in increments of 1 L, The force is σ into p Η of hydrochloric acid, and ρ Η is 5.5. The final concentration of the probe D Ν 0 is 0. 0 3nmo 1 / L > and, in order to make the carboxylic acid on the surface of the carrier and the probe 312XP/ Inventive manual (supplement) /94_08/94丨08S98 In addition to loading, setting, setting the convex part and the flat part of the 6 convex parts on the flat part, the aqueous solution solution of the concave central part, "NA ° as the storage PBS ( The 胺·2g adjustment was carried out by condensing the amino group at the end of 31 1354792 of the DNA, and adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to make The final concentration was made 50 mg/mL. Then, the mixed solution was spotted on the carrier convex portion with a glass capillary. Then, the carrier was placed in a closed plastic container at 37°匚, humidity 100%. The mixture was allowed to stand for about 20 hours and then washed with pure water. The reaction scheme is shown in Fig. 7. (Adjustment of sample DNA) As the sample DNA, the system was made. The DNA having the SEQ ID NO: 4 of the base sequence which can be hybridized to the probe DNA immobilized on the DNA-immobilized vector is 968. The adjustment method is as follows. The DNA of SEQ ID NO: 2 and SEQ ID NO: 3 is synthesized. Dissolve it in pure water to make the concentration 1 〇〇; /Μ. Then, prepare PKF3 plastid DNACTAKARA BI 0 (stock), product number: 3 1 0 0 ) (SEQ ID NO: 5: 2 2 6 4 bases ), using it as a template (temp 1 ate ), using DNA of SEQ ID NO: 2 and SEQ ID NO: 3 as a primer, and performing amplification by a PCR reaction (Polymerase Chain Reaction)

The conditions of P C R are as follows. That is, add EXT aq 2 // L, 1 0 X ExBuf f er40 μ. L > dNTP Mix 32/z L (above is TAKARA BIO (share), attached to article number RR 0 0 1 A ), sequence No. 2 solution 2 # L, SEQ ID NO: 3 solution 2 L, template (sequence number 5) 0 · 2 // L, in pure water increments to a total of 4 0 0 # L. These mixed solutions were divided into 4 microtubes, and a PCR reaction was carried out using a thermal cycler. It was purified by ethanol precipitation and dissolved in 40 μL of pure water. A part of the solution after the PCR reaction was confirmed by electrophoresis, and it was confirmed that the amplified base 32 312XP/invention specification (supplement)/94_08/94108898 1354792 has a base length of about 690 bases. Enlargement of sequence number 4 (9 6 8 bases). Then, a 9-base random primer (TAKARA BIO (manufactured by TAKARA BIO), product number: 3802) was dissolved to a concentration of 6 mg/mL, and 2 / 8 L was added to the above-mentioned PCR reaction, and the purified DNA solution was added. in. After heating the solution to 100 ° C, it was rapidly cooled on ice. Add the buffer 5 " L, d NTP mixture attached to K 1 e η 〇w Fragment CTAKARA BIO (product number 2140ΑΚ) (the concentrations of d ATP, d TTP, and d GTP are respectively 2.51^, (3 (: Ding 卩 concentration is 400 / ^ 1 〇 2.5; ^ 1 ^. Then join

Cy3-dCTP (manufactured by Amersham phamac i a Biotech; product number PA53021) 2/iL. To this solution, 10 U of Klenow Fragment was added, and the mixture was allowed to stand at 37 ° C for 20 hours to obtain a sample DNA identified by Cy3. Further, since random primers are used in the identification, the length of the sample DNA is uneven. The longest sample D N A is SEQ ID NO: 4 (9.66 bases). The solution of the sample DNA was taken out and confirmed by electrophoresis. As a result, the strongest band appeared in the vicinity of the base of 690 I, and the region corresponding to the base shorter than the short base was thinly dispersed. The state of (smear). Then, it was purified by ethanol sedimentation and dried. The labeled sample DNA was dissolved to 1% by weight of BSA (bovine serum albumin), 5xSSC (5xSSC refers to 20xSSC (manufactured by Sigma) diluted 4 times with pure water. Also '20xSSC with pure water Diluted to 2 times is expressed as lOxSSC, 2x dilution of 20xSSC is expressed as i〇xsSC, 100 times release is expressed as 0.2xSSO), 0.1% by weight of SDS (sodium dodecyl sulfate), 0 · 0 A solution of 1% by weight of salmon sperm DNA (each concentration is the final concentration) 33 312XP / invention specification (supplement) / 94-08/94108898 1354792 into 4 Ο 0 / i L, as a stock solution for hybridization. In the following examples and comparative examples, the sample solution at the time of hybridization is 1% by weight of BSA '5xSSC '0.0 1% by weight of salmon sperm DNA, 0.1% by weight, unless otherwise limited. The solution of SDS (each concentration is the final concentration) is diluted to 200 times. Further, the concentration of the sample D N A of the solution was measured and found to be 1.5 n g / /z L . (Modification of glass beads) 10 g of glass beads having a diameter of 150 k / m was dipped into 10 N N a Ο Η solution # and washed with pure water. Next, APS (3-aminopropyltriethoxydecane; manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in pure water at a ratio of 2% by weight, and then the glass beads were immersed for 1 hour, and taken out from the solution. After drying at 110 ° C for 10 minutes. Thus, the amine group is introduced to the surface of the glass beads. Then, 5.5 g of succinic anhydride was dissolved in 1 -mercapto-2-pyrrolidone 3 3 5 m L. Add the above-mentioned succinic acid solution to 1M of 50 mL of succinic acid sodium (addition of boric acid 3.09 g and p Η adjusted sodium hydroxide, and pure water to 50 m L; ρ Η is 8. 0). The above glass beads were immersed in the mixture for 20 minutes. After impregnation, it is washed with pure water and dried. Thus, the amine group on the surface of the glass bead is reacted with succinic anhydride to introduce a carboxyl group onto the surface of the glass bead. (Hybridization) The sample DNA was hybridized on the vector to which the probe DNA was immobilized as described above. Specifically, the carrier nucleic acid prepared in advance is fixed to the carrier of the convex portion, 50//L of the hybridization solution is dropped, and the modified glass beads 2 mg are mixed in the concave portion of the carrier, and the glass cover is covered thereon. sheet. The glass cover sheet is sealed with a paper tape so that the hybridized solution does not dry. 34 312XP/Invention Manual (Supplement)/94-08/94] 08898 1354792 Dry. In the glass cover sheet surface, among the four sides, the two opposite sides are formed by using a photoresist having a thickness of 8/m and a width of 1 mm formed by photolithography. Thus, at the time of hybridization, the distance (pitch) of the carrier convex portion and the cover glass can be made 8/z m. It is fixed in a plastic container provided on a rotating surface of a microtube rotator (product code: 1-4096-01, manufactured by Azwang Corporation) at a humidity of 100% at 60 °C. Allow to stand for 10 hours under conditions. At this time, the number of rotations of the rotating machine is set to 3 r p m , and the rotating surface of the rotating machine is made to be at right angles to the horizontal plane. Further, the probe DNA fixing surface φ of the carrier is formed at a right angle to the rotating surface of the rotating machine. After standing, the cover glass was peeled off from the carrier, and the carrier was washed and dried. (Measurement) The carrier after the above treatment was placed in a DNA crystal scanner (GenePix 4000B of Axon Instruments), and the measurement was performed with the laser output 33%' photomultiplier voltage set to 500. . The results are shown in Table 1. Here, the "fluorescence intensity" means the average value of the fluorescence intensity in the focus (s ρ 〇 t).

Further, in the present embodiment, glass beads were used, but even if ceramic beads or Teflon (registered trademark) beads were used, substantially the same results as in Table 1 were obtained. (Comparative Example 1) An experiment was carried out when no glass beads were placed. The experimental procedure was carried out in the same manner as in Example 1 except that the glass beads were not mixed at the time of the hybridization. The results are shown in Table 1. It was confirmed that the fluorescence intensity was lower than that of Example 1. Further, the fluorescence intensity distribution with respect to the carrier convex portion of Comparative Example 1 was not uniform (sweet 35 312 XP / invention specification (supplement) / 94-08/94108898 1354792 donut-like distribution), the result of Example 1, The distribution of the fluorescence intensity of the convex portion of the carrier is substantially uniform. (Comparative Example 2) An experiment was carried out in the case where a flat PMMA carrier having no uneven portion was provided. The experimental procedure was performed except that (1) a flat carrier was used; (2) the point of the probe DNA was carried out using a dedicated machine (manufactured by Nippon Laser Electronics Co., Ltd., GeneStamp II); (3) and, in the case of a glass cover sheet. 4 sides are adhered to a polyester film having a thickness of 20 〇Aim and a width of 1 mm, and a gap can be set between the carrier and the cover glass φ in such a manner that the beads can be stirred, and the beads and the sample solution are mixed and hybridized in the gap. In addition, the same procedure as in Example 1 was carried out. The results are shown in Table 1. It can be seen that the fluorescence intensity is lower than that of the first embodiment. Further, the damage of the point (s ρ 〇 t) not seen in the embodiment 1 can be confirmed. It is presumed that when the hybridization is carried out, the beads injure the probe immobilization surface. Further, the diameter of the beads was set to 1 " m, and the experiment was carried out in this comparative example, and the fluorescence intensity was lower, which was about 1 500. It is presumed that this is due to the resistance of the hybrid solution, and it is confirmed that the beads are difficult to move.

(Example 2) An experiment using a stirring effect of a bubble was performed. The experimental procedure was the same as in Example 1 except that the cover glass cover of the hybrid step was fed with a bubble of 0.9//L in a micro syringe and no glass beads were added. Further, the carrier is fixed and rotated so that the rotating surface of the rotating machine tilted in the vertical direction is parallel to the probe fixing surface of the carrier, so that the bubble moves only around the sealed sample solution. (Embodiment 3) 36 312 ΧΡ / invention manual (supplement) / 94-08/94108898

1354792 The same experiment as in Comparative Example 2 was carried out using a container for holding a solution of the cross-sectional structure shown in Fig. 2, and a glass cover sheet. That is, the uneven portion is not provided on the carrier but is provided on the cover glass. The positional relationship between the container and the flat PMMA carrier is carefully matched as shown in Fig. 2. If the beads are moved while being hybridized, the distance between the solidified surface of the probe DNA and the container 4 holding the solution is Since the diameter of the beads 2 is small, the glass beads do not come into contact with the probe DNA fixing surface 1 and the glass beads can be moved. The results are shown in Table 1. Regarding the fluorescence intensity, the same results as in the first embodiment can be obtained. If the results of Comparative Example 2 are combined, it is considered important that the beads do not contact the probe face. In the method of the present embodiment, it is preferable to accurately position the convex portion of the cover sheet and the portion to which the probe DNA is fixed. (Comparative Example 3) An experiment was carried out in the case where a flat PMMA carrier having no uneven portion was provided and was not stirred by beads. The same operation and measurement results as in Comparative Example 2 were carried out except that the beads were not mixed in the hybridization solution and rotation was not performed, as shown in Table 1. [Table 1] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Target concentration (ng/μ. L) 1. 5 1.5 1. 5 1.5 1. 5 1. 5 Substrate shape uneven slab Concave flat plate gap (// m) 8 8 8 8 200 8 Stirring method Beads Bubble beads No beads No rotation with or without fluorescence intensity 1 2000 8800 11800 2900 3900 700 Noise 45 50 300 50 300 300 312XP/Invention Manual (Supplement)/94-08/94108898 Substituted 〇定玻璃结固固重粒 and 37 1354792 (Example 4) In the glass beads of Example, five experiments were selected. Experimental procedure In the same manner as in Example 1, the diameter of the glass beads was 10, 20, 50, 1 0 0, 2 Ο Ο μ m. The results are shown in Table 2. (Comparative Example 4) An experiment was carried out in the same manner as in Example 1 except that the diameter of the glass beads was set to 300 / im, 400 " m. The results are shown in Table 2. [Table 2] Example 4 Comparative Example 4 Size (V m ) 10 20 50 1 00 2 0 0 300 400 Fluorescence intensity 8 2 0 0 1 0 5 0 0 1 2 7 0 0 1 2 0 0 0 1 2 5 0 0 3100 3 0 0 0

From the above, the use of beads of 10 to 2 0 0 // m clearly showed a stirring effect, while the beads of Comparative Example 4 of 300° and 4 0 0 #m failed to obtain a remarkable effect. The reason is that since the distance between the concave portion of the carrier and the cover glass is 208 " m, the beads that are barely set to 300, 4 0 0 # m are sandwiched by the cover glass and the carrier, so that the distance cannot be moved. The reason. According to the examples and comparative examples, it is understood that the fluorescence intensity is weak when the beads cannot move. Further, as is clear from Example 4, the preferred size of the beads is 10 // m or more, and more preferably 2 Ο μ m or more. (Example 5) The same experiment as in Example 1 was carried out using a carrier having the characteristics described later. In the center of the carrier, a concave portion having a diameter of 10 m and a depth of 0·3 m is provided, and among the concave portions, a crotch portion having a diameter of 0.2 mm and a height of 0.3 mm at 64 (8 x 8) is provided. The characteristics of the other vectors and the experimental procedures are the same as in the first embodiment. Further, the diameter of the glass beads is set to 10, 2 0, 5 0, 1 0 0 ' 2 0 0, 38 312 ΧΡ / invention specification (supplement) / 94-08/94108898 1354792 3 Ο Ο // m. The results are shown in Table 3. (Comparative Example 5) An experiment was carried out in the same manner as in Example 5 except that the diameter of the glass beads was changed to 4 0 0 // m. The results are shown in Table 3. [Table 3] Example 5 Comparative Example 5 Size (/im) 10 20 50 10 0 200 300 400 Fluorescence intensity 88 0 0 11000 1 3 0 0 0 1 2 0 0 0 1 2 7 0 0 1 2 0 0 0 2 7 0 0

If the beads of 1 0 ~ 3 0 0 # m are used, the stirring effect can be clearly seen, and with the beads of 400 k / z m, no significant effect can be obtained. The reason is that since the distance between the concave portion of the carrier and the cover glass is 308/zm, the beads of 400 m are barely placed, and the cover glass is caught by the carrier, so that it cannot be moved. From the examples and comparative examples, it is understood that the fluorescence intensity is weak when the beads cannot move. Further, as is clear from Example 5, the preferred size of the beads is 10 // m or more, and more preferably 2 0 // m or more. (Example 6) An experiment was conducted in the case where the heights of the convex portions were different. The convex portion of the injection molded article of PMMA used in Example 1 was ground with rubbing paper so that the height of the convex portion was different. That is, the carrier (carrier A) having a convex portion (four places) lower than the other convex portions (the convex portion as the reference) by 30/m is formed separately; it is 50"m lower than the other convex portions. Carrier (carrier B) of the convex portion (4 places). Further, the difference between the height of the convex portion (the convex portion as the reference) and the height of the flat portion other than the lower portion of the carrier is 3 # m or less. The adjustment of the spotted probe D N A was carried out in the same manner as in the first embodiment. Then, probe DNA was carried out in the same manner as in Example 1 at four points on the upper surface of the convex portion as the reference and on the lower convex portion 39 312XP/invention specification (supplement)/94-08/94108898 1354792. In the same manner as in the first embodiment, the adjustment of the sample DNA for hybridization was carried out. Hybridization and measurement were also carried out in the same manner as in Example 1. The average value of the fluorescence intensity on the convex portion on the basis of the reference and the average value of the fluorescence intensity on the convex portion on the lower side are shown in Table 4.

[Table 4] Example 6 Carrier A Carrier B As a reference, a convex portion having a lower portion of 3 0 // m is used as a reference. A convex portion having a lower convex portion of 5 0 μm II 1 3 0 0 0 1 2 0 0 0 1 2 6 0 0 8 9 0 0 As described above, even when the height of the convex portion is uneven (50 0 m or less), the fluorescence intensity equivalent to that of the first and second embodiments can be obtained. (Example 7) A review was made on the case where the upper surface of the convex portion differs from the flat portion. The flat portion of the injection-molded article of Ρ Μ A used in Example 1 was sanded, and a carrier (carrier C) having a height difference of 30/m between the upper surface of the flat portion and the upper surface of the convex portion was prepared, and the height difference was 2 types of 50//m carrier (carrier D). That is, the carrier C has a height of the convex portion which is 30#m higher than the height of the flat portion. Experimental procedure In the same manner as in Example 1, the alignment of the probe D N A was applied, the probe DNA solution on the convex portion was spotted, the sample DNA was adjusted, and the glass beads were modified. Hybridization was carried out by substituting the crepe sheet (thickness 60 m) for the formation of a polymer on the cover glass in Example 1. Further, (S ρ 〇 t) D N A solution was placed at four points on the convex portions of the respective carriers. Then, the average value of the fluorescence intensity of the spot (four places) where D N A is clicked is obtained. The results are shown in Table 5. 40

312XP/Invention Manual (Supplement)/94-08/941OS89S 1354792 [Table 5] Example 7 Carrier C Carrier D Fluorescence intensity 11500 8 7 0 0 It is thus known that even if the height of the flat portion is different from the height above the convex portion In the case of (50 0 m or less), the same fluorescence intensity as in Example 1 can be obtained. (Example 8)

The substrate in which the glass cover sheet was sealed with a paper tape in Example 1 was placed in a Vortex (manufactured by Scientific Industries, Inc.), and the glass beads were moved by vibration to carry out hybridization stirring. The procedure was carried out in the same manner as in Example 1 except for the vortex mixer. The results are shown in Table 6. Shows strong fluorescence intensity. (Example 9)

The magnetic beads were mixed at the time of hybridization, and the magnetic beads were moved by changing the surrounding magnetic field to carry out an experiment of stirring the hybridization solution. First, make a machine that moves back and forth as shown in Figure 8. The experimental procedure was (production of DNA immobilization carrier) (fixation of probe D N A) (adjustment of sample D N A ) (measurement) was carried out in the same manner as in Example 1. The hybrid system mixes 1 mg of magnetic beads (made by Treyal) made of 50# m in diameter instead of glass beads in the concave portion of the carrier, and is placed in the above-mentioned self-made machine instead of the rotator. Other than that, it was carried out in the same manner as in Example 1. The results are shown in Table 6. Shows strong fluorescence intensity. (Comparative Example 6) 41 312XP/Invention Manual (Supplement)/94-08/94] 08898 1354792 The same experiment as in Example 9 was carried out using a flat PMMA carrier which was not provided with uneven portions. Experimental procedure, except (1) using a flat carrier; (2) using a dedicated machine (made by Japan Laser Electronics Co., Ltd., GeneStamp II) to probe DNA; (3) and, in the cover glass 4 A polyester film having a thickness of 200//m and a width of 1 mm is adhered to each other, and a gap is provided between the carrier and the cover glass in such a manner that the magnetic beads can be stirred, and the magnetic beads and the sample solution are made in the gap. Hybridization was carried out by mixing; (4) The self-made machine shown in Fig. 8 was placed in the same manner as in Example 1 except that the cover glass was faced downward. By bringing the glass Φ cover sheet face down, the magnetic beads can be attracted to the cover glass surface, and the solution can be agitated without being in contact with the opposite probe attachment surface. The results are shown in Table 6. Only the fluorescence intensity which is inferior to that of Example 9 was obtained. Further, the damage of the point (s ρ 〇 t) which was not observed in the ninth embodiment was confirmed. In Comparative Example 6, the magnetic beads which were attracted by the magnet were agglomerated at a width of 200 μm which was set by the polyester film, and since they were moved in a state of blocking, the block was The object will come into contact with the probe fixing surface. [Table 6] Example 8 Example 9 Comparative Example 6 Bead type glass magnetic body magnetic force external force vibration magnetic force fluorescent intensity 9 9 0 0 7 8 0 0 2 8 0 0 10) (Example except bead use The stability and stability of the Yttria-stabilized Zirconia (for zirconia in a ratio of 2.5 mole % mixed with antimony trioxide) made of a diameter of 1 2 5 / m, and examples 1 Experiment was carried out in the same manner. The result was that the fluorescence intensity after hybridization was approximately the same as in the case of 42 3 ] 2XP / invention specification (supplement) / 94-08/94108898 1354792. However, the mixed beads were 2 mg even for The disturbance of the solution when covering the cover glass, the beads are difficult to move, so it is easy to place. The reason is that the specific gravity of the oxidized beads is 6.05 g/cm3, which is close to 3 times the specific gravity of the glass. (Example 1 1 ) The same experiment as in Example 1 was carried out using the concentration of the sample DNA adjusted to 0.73, 0.29, and 0.15 ng//zL. The results are shown in Fig. 9. In Fig. 9, the example 1 is described together. The results of Comparative Example 1. 瘫 (Comparative Example 7) The concentration of the sample DNA was adjusted to The same experiment as in Comparative Example 1 was carried out at 0.73, 0.29, and 0.15 ng//zL. The results are shown in Fig. 9. The results of Example 1 and Comparative Example 1 are collectively shown in Fig. 9. Thus, the condition of 4 conditions is examined. The body concentration was confirmed to have an effect of stirring with beads. (Example 1 2) A test for SNP (single nucleotide polymorphism) by DNA wafer was carried out. The experimental procedure was (DNA immobilization vector). (production) (modulation of sample DNA) (modification of glass beads) (hybridization) (measurement) was carried out in the same manner as in Example 1. The concentration of the sample DNA was 1 · 5 ng / " L. Among them, hybridization This was carried out at 42 ° C. The probe DNA was synthesized by synthesizing the 5'-endylated DNA of SEQ ID NO: 6 and SEQ ID NO: 7. The SEQ ID NO: 6 and 7 differed by only one base. The sequence of 10 bases from the 5' side is not complementary to the sample DNA, and the portion excluding the portion of SEQ ID NO: 6 (20 bases) is completely complementary to the sample DNA. 43 312XP /Invention Manual (supplement)/94-08/94108S98 1354792 These two types of DNA are implemented and implemented The same procedure as in Example 1 was carried out on the carrier convex portion. The results are shown in Table 7. By the method of the present invention, the difference of one base of the two types of probe DNA can be detected. [Example 2 Example 1 2 SEQ ID NO: 6 7 Fluorescence intensity 1 0 5 0 0 3 2 0 0 (industrial availability)

According to the present invention, it is possible to provide a stirring method which promotes the reaction of the selective binding substance immobilized on the carrier with the test substance, and a good signal intensity and S / N ratio can be obtained even in a trace amount of the sample. That is, by using the stirring method of the present invention, the signal intensity and the S/N ratio (i.e., the sensitivity) of the selective immobilization carrier represented by the DNA wafer can be improved, even for a small amount of clinical samples. Can also be measured. According to the present invention, diagnosis and examination can be carried out by using a clinical site in which a binding substance is selected as a representative of a D N A wafer. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic cross-sectional view showing an embodiment of the present invention. Figure 2 is a schematic cross-sectional view showing an embodiment of the present invention. Figure 3 is a schematic illustration of a carrier. Figure 4 is a schematic cross-sectional view of the carrier. Fig. 5 is an example of a DNA wafer holding jig. Fig. 6 is a schematic view showing a case where a carrier is used as a support layer/selective substance-immobilized layer. 44 312XP/Invention Manual (supplement)/94-08/9410S898 1354792 Figure 7 is a reaction flow diagram for the selection of binding substances on the surface of Ρ Μ Μ A. Figure 8 is a schematic view of a jig used in Example 9. Figure 9 is the fluorescence intensity in the case where the target (t a r g e t) concentration is changed. [Main element. Description of symbols] 1 Selected binding substance (DNA) fixed to the carrier 2 Microparticles (beads) 3 Carrier φ 4 Container 11 holding the reaction solution Flat portion 1 2 Concave and convex portion 13 D Ν Α Wafer 14 Objective lens 15 Ray The excitation light 16 is used to hold the microarray against the spring of the clamp. 3 1 Select the bonding substance immobilization layer ^ 32 Support layer

41 PMMA

42 DNA 51 Magnet 5 2 The direction of the back and forth movement of the magnet 53 Substrate 45 312XP/Invention Manual (supplement)/94-08/9410SS98 1354792 Sequence Listing <110>Tosoh Corporation<120> Solution Dad Mixing Τ·δ Method <130> Case No. 04080 060) 7 <170> Patent In Ver. 2. 1 <210> 1 <2Π> 60 <212> DNA <213> Plasmid pKF3 <400> 1 acattttgag gcatttcagt Cagttgctca atgtacctat aaccagaccg ttcatctgga 60 <210> 2 <zn>2〇<212> DNA ^

<213> Plasmid pKF3 <400> Z gggcgaagaa gttgtccata 20 <Z10> 3 <210 20 <212> DNA <213> Plasmid pKF3 <400> 3 gcagagcgag gtatgtaggc 20

<Z10> 4 <2Π> 968 <212> DNA <213> Plasmid pKF3 <400> 4 gggcgaagaa gttgtccata ttagccacgt ttaaatcaaa actggtgaaa ctcacccagg 60 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt 120

caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt 180 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt 240 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacgaaat tccgtatgag 300 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct 360 ttacggtclt Shu aaaaaggcc gtaatatcca gatgaacggt ctggttatag gtacattgag 420 caactgactg aaatgcctca aaatgttctt Shu acgatgcca ttgggatata tcaatggtgg 480 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact 540 caaaaaatac gcccggtagt eatcttattt cattatggtg aaagttggaa cctcttacgt 600 gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga 660 caccaggatt tatttattct gcgaagtgat cttccgttcg acggagttcc actgagcgtc 720 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 780 312XP / manual (complement member) Inventive / 94-〇S / 94108898 46 1354792 ctgcttgcaa aca2saaaac caccgctacc agcggtggtt丨gtttgccgg atcaagagct 840 accaactctt tltccgaagg taactggctt cagcagagcg cagataccaa at2ctgtcct 900 tctagtgtag ccgtagtUg gccac Cactt caagaactct gtagcaccgc ctacatacct 960 cgctctgc 968 <210> 5 <211> 2246 <212> DNA <213> Plasmid pKF3 <400> 5 atggcaacag tcaatcagct ggttcgaaag ccgcgagctc gtaa.agtggc caaatclasc 60 gttccggctc tcgaggcatg cccgtagaag cgtggcata丨gcacacgtgt atacactact 120 actccgaags aaccgaattc agcectgcgc aagctttgcc gCEtacgcct gaccaacggt 180 ttcgaggtca cctcatatat aggtggtgaa ggacacaacc tgcaggaaca ctcigttatc 240 ctgatcagag gcggccgcgt laaagatctg cccgggatcc ggtaccacac cgtccgcggc 300 gctctagsct gctccggagt aaaggaccgt cgacaggatc eatcgaaata cgetgtaaaa 360 cgtccgaagg cctaatagaa gctagcttgg cactgggcca agctgaattt ctgccattca 420

tccgcttatt atcacttstt caggcgtagc accaggcett taagggcacc aataacigcc 480 ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat 540 tctgccgaca tggaagccat cacagacggc atgatgaacc tgaatcgcca gcggcatcag .600 caccttgtcg ccttgcgtat aatatttgcc catagtgaaa acgggggcga agaagttgtc 660 catattagcc acgtttaaat caaaactggt gaaactcacc cagggattgg cteagacgaa 720 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac 780 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga 840 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggteaacac tatcccatat 900 caccagctca ccgtctttca ttgccatacg aaattccgta tgagcattca tcaggcgggc 960 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa Ί020 ggccgtaata tccagatgaa cggtdggtt ataggtacat tgagcaactg actgaaatgc 1080 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt 1140 tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg 1200 tagtgatctt alttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca 1260 ttttcgccaa Aag ttggccc agggcttccc ggtatcaaca gggacaccag gatttattta 1320 ttctgcgaag tgatcttccg ttcgacggag ttccactgag cgtcagaccc cgtagaaaag 1380 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 1440 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 1500 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 1560

ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcectct gctaatcctg 1620 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 1680 tagttaccgg ataaegcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 1740 ttggaecgaa cgacctacac cgaactgaga tacctacagc etgagcattg agaaagcgcc 1800 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 1860 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 1920 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 1980 aaaaacgcca gcaacgcggc ctttttacgg ttcctEECCt tttgctggcc ttttgctcac 2040 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 2100 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 2160 gaagaaecat tctgaaatga gctgttgaca attaatcatc gaactagtta actagtacgc 2220 aagttcacgt aaaaaggeta tcgacc 2246

312XP / invention manual (supplement) like -08/941OS89S 47 1354792

<210> 6 <211> 30 <212> DNA <213> Plasmid pKF3 <400> 6 tttttttttt acattttgag gcatttcagt

<210> 7 <211> 30 <212> DNA <2Ί3> Plasmid pKF3 <400> 7 tttttttttt acattttgag acatttcagt 3 ] 2XP/invention specification (supplement)/94-08/94108898

Claims (1)

AUG 1 〇2011 Replacement of this 1354792 X. Patent application scope: 1. A solution stirring method for contacting a selective binding substance immobilized on a surface of a carrier with a solution containing a substance to be tested which reacts with the selected binding substance, a method of stirring the solution; characterized in that a carrier having a structure in which the microparticles or the bubble are not in contact with the fixing surface of the selective binding substance is used, and the microparticle or the bubble is mixed in the solution containing the test substance to move the microparticle or the bubble without The fixed surface contact of the binding substance is selected. 2. The solution stirring method according to claim 1, wherein the carrier in which the fine particles or the bubbles are not in contact with the fixing surface of the bonding material is provided with a concave-convex portion and the selective bonding substance is fixed on the convex portion. Carrier. 3. The method of stirring a solution according to claim 1 or 2, wherein the solution is stirred by moving the fine particles. 4. The method of stirring a solution according to claim 1 or 2, wherein a container for holding the solution is used. 5. The method of stirring a solution according to claim 1 or 2, wherein the solution is stirred by moving the fine particles, and the concave and convex portions are provided on the carrier; the binding substance is selected to be fixed on the convex portion, and the fine particles are attached to the concave portion. mobile. 6. The solution stirring method according to claim 1 or 2, wherein the carrier is provided with a flat portion and a concave portion, and the bonding substance is selected to be fixed on the plurality of convex portions, and the height of the convex portion is substantially the same And the difference in height between the flat portion and the convex portion is 5 0 " m or less. 7. The method of stirring a solution according to claim 1 or 2, wherein the 49 94108898 1354792 microparticles are moved by any one of gravity, magnetic force, vibration of the carrier or a combination thereof. 8. The method of stirring a solution according to claim 1 or 2, wherein the binding substance is selected as a nucleic acid. 9. The method of stirring a solution according to claim 1 or 2, wherein the selective binding substance is reacted with the test substance. The solution agitating method according to claim 5, wherein the maximum width of the microparticles is 10/zm or more, and the height difference between the upper surface of the carrier convex portion and the concave portion is lower. 11. A method of stirring a solution by contacting a selective binding substance immobilized on a surface of a carrier with a solution containing a substance to be tested which reacts with the selective binding substance to agitate the solution; characterized by using microparticles or The container in which the bubble does not contact the structure in which the binding substance is fixed is contacted, and the microparticles or bubbles are mixed in the solution containing the test substance to move the microparticles or the bubble without being in contact with the fixing surface of the selective binding substance. 1 2. The solution stirring method according to the first aspect of the invention, wherein the container of the holding solution having the structure in which the micro φ particles or the bubbles are not in contact with the fixing surface of the bonding material is provided, and the container for holding the solution having the uneven portion is provided. And the binding substance is selected to be fixed to the surface of the carrier below the convex portion. 1 3. A solution agitation method according to claim 11 or 12, wherein the solution is disturbed by moving the microparticles. 1 4. The solution stirring method according to claim 11 or 12, wherein the solution is stirred by moving the microparticles, and the minimum width of the microparticles is between the fixed surface of the selective binding substance and the container holding the solution. The shortest distance is greater. 50 94108898 1354792 15. The solution agitating method according to claim 11 or 12, wherein the particles are moved by any one of gravity, magnetic force, vibration of the carrier or the group 1 of the carrier. 1 6. In the solution stirring method of claim 11 or 12, the binding substance is selected as a nucleic acid. 17. In the solution stirring method of claim 11 or 12, the selective binding substance is reacted with the test substance. 18. A method of stirring a solution, wherein a binding substance is selected on a convex portion fixed to a carrier, and a solution containing a substance capable of reacting with the selective binding substance is contacted, and the solution containing the substance to be tested is mixed with the particles. The particles are moved to agitate the solution; characterized in that a container of the solution is used, and the minimum width of the particles is greater than the shortest distance between the face of the selected binding substance and the container holding the solution. 1 9. The solution stirring method according to claim 18, wherein the solution is stirred by moving the microparticles, and the minimum width of the microparticles is larger than the shortest distance between the fixing surface of the binding substance and the container holding the solution. The solution stirring method of claim 18, wherein the solution is stirred by moving the fine particles, and the concave and convex portions are provided on the carrier; the bonded material is fixed on the convex portion, and the fine particles are attached to the concave portion. mobile. The solution stirring method of claim 18, wherein the carrier is provided with a flat portion and a concave-convex portion, and the bonding substance is selected to be fixed on a plurality of convex portions, and the height of the convex portion is substantially the same. Further, the height difference between the flat portion and the convex portion is 50 μm or less. 94108898 its) the quilt is fixed by the continuation of the 51 51 rI354792 22. The solution stirring method of claim 18, wherein either gravity, magnetic force, vibration of the carrier or this The combination of the particles causes the particles to move. 23. The method of stirring a solution according to claim 18, wherein the binding substance is selected as a nucleic acid. 24. The method of stirring a solution according to claim 18, wherein the selective binding substance is reacted with the test substance. The solution agitating method according to claim 20, wherein the maximum width of the microparticles is ΙΟμηη or more, and the height difference between the upper surface of the carrier convex portion and the concave portion is lower. 52 94108898 1354792 如 6 1 Ο _ Replacement page Η—, Drawing: 94108898 53